Annual Cycle and Diversity of Species and Infraspecific Taxa of Ceratium (Dinophyceae) in the Ligurian Sea, Northwest Mediterranean1

Home , Ceratium, Cyclosalpa, Cyclosalpa bakeri, Dinophyceae, Dolioletta gegenbauri, Salpa fusiformis

J. Phycol. 43, 1149–1163 (2007) 2007 Phycological Society of America DOI: 10.1111/j.1529-8817.2007.00417.x

ANNUAL CYCLE AND DIVERSITY OF SPECIES AND INFRASPECIFIC TAXA OF CERATIUM (DINOPHYCEAE) IN THE LIGURIAN SEA, NORTHWEST MEDITERRANEAN1

Alina Tunin-Ley, Jean-Philippe Labat, Ste´phane Gasparini, Laure Mousseau, and Rodolphe Lemeé2 Laboratoire d’Oceánographie de Villefranche, Universite´ Pierre et Marie Curie-Paris 6, 06230 Villefranche-sur-Mer, France CNRS, Marine Microbial Ecology Group, Laboratoire d’Oceánographie de Villefranche, 06230 Villefranche-sur-Mer, France

We examined the well-documented and species- on. The first monograph was published in the rich dinoflagellate genus Ceratium Schrank in the beginning of the 20th century (Jørgensen 1911) fol- northwest Mediterranean Sea as a possible model lowed by many other taxonomic studies. Ceratium for marine phytoplankton diversity and as a bio- ranges from polar to tropical waters, in open sea as logical indicator of global climate change. First, we well as in neritic areas (Sournia 1967, Dodge and investigated the influence of counting effort; we Marshall 1994). Since it often represents a signifi- then documented temporal changes in Ceratium spe- cant part of the microphytoplankton in terms of cific and infraspecific taxa over 2 years (2002 and abundance and diversity, especially within the the- 2003) in the Villefranche Bay based on a monthly cate dinoflagellates, it plays a significant role in bio- net sampling. Finally, we tried to identify factors geochemical cycles. Furthermore, some species of associated with shifts in biodiversity. The calculation Ceratium are known to form red-tide blooms (Bocks- of taxonomic diversity, regularity, and richness were tahler and Coats 1993). The genus is very species highly dependent on counting effort. We deter- rich, with many species described from tropical and mined that a minimal sample volume of 70 L was subtropical areas (Dodge and Marshall 1994). A needed to obtain a good estimation of species rich- considerable amount of ecophysiological data exists ness. The annual cycle was characterized by a sea- as some common Ceratium species have been used sonal trend of high winter species richness followed in experimental and in situ studies ranging from by low spring biodiversity. Infraspecific variability investigations of bioluminescence (Sullivan and not only appeared to depend on water temperature Swift 1995) to cell division and growth rates but also seemed to be influenced by bottom-up con- (Elbra¨chter 1973, Weiler and Eppley 1979, Weiler trol and was strongly affected by top-down control. 1980), cellular functioning (Sato et al. 2004), tro- Thus, the occurrence of high concentrations of phic relationships (Smalley et al. 1999, 2003, Skovg- salps (Thalia democratica) and copepods larger than aard et al. 2000), and taxonomy (Sournia 1967). 2mm(Calanus helgolandicus) coincided with a dras- The genus has been also used as a biological indica- tic decrease of Ceratium abundance and diversity tor of water masses in the North Atlantic Ocean during spring 2003. Ceratium is sensitive to both abi- (Dodge and Marshall 1994, Raine et al. 2002), in otic and biotic factors and could prove to be a good the Pacific Ocean (Dodge 1993, Sanchez et al. candidate as a biological indicator of global change. 2000), in the Mediterranean Sea (Dowidar 1973), and in the Arctic Ocean (Okolodkov 1996). How- Key index words: annual cycle; Ceratium; dinofla- ever, like many planktonic groups, little is known gellates; diversity; infraspecific variability; Medi- about factors influencing the biodiversity of this terranean Sea; predation pressure genus, although it is characterized by a large num- Abbreviations: BIOSOPE, Biogeochemistry and ber of species and infraspecific taxa. While some Optics South Pacific Experiment; CA, correspon- taxonomic and biogeographical studies have tried dence analysis; CNRS, Centre National de la to assess the species richness of Ceratium in particu- Recherche Scientifique; CPR, continuous plank- lar areas (Sournia 1967, Dodge 1993, Semina and ton recorder; CTD, conductivity-temperature- Levashova 1993, Dodge and Marshall 1994), there is density; DOM, dissolved organic matter very little information on temporal patterns and the dynamics of biodiversity at annual and interannual timescales for Ceratium, particularly in terms of In the Mediterranean Sea, the genus of armored infraspecific variability. dinoflagellates, Ceratium, a component of the micro- In this study, we first focused on the validation of phytoplankton, was described and studied very early an adequate methodological approach for studying the genus Ceratium in terms of occurrence and biodiversity; more particularly, we tried to deter- 1Received 23 October 2006. Accepted 14 May 2007. mine the minimal volume of water to analyze to 2Author for correspondence: e-mail [email protected]. obtain reliable biodiversity parameters. Second, we

1149 1150 ALINA TUNIN-LEY ET AL. described the annual variations of specific and infra- Identification and counting were completed to the infraspe- specific composition through 2 years of monthly cific level, using standard taxonomic studies (Gourret 1883, sampling in the Villefranche Bay. Third, we investi- Jørgensen 1911, 1920, Tre´gouboff and Rose 1957a,b, Halim 1960, Dodge 1982, Steidinger and Tangen 1997) and the gated the relationships between abiotic and biotic nomenclature established by Sournia (1967) as a reference. factors on population dynamics in Ceratium. The taxonomic authorities are presented in Table 1. All the infraspecific taxa and species of the genus Ceratium that are MATERIALS AND METHODS cited in the present study are illustrated in the work of Sournia (1967), and, if not, references for corresponding descriptions Study site and sampling. Sampling was conducted at Point B are given in Sournia (1967). For standardization, the same in the Villefranche Bay (Fig. 1), a long-term monitoring site. person counted all the samples. The station is situated in the mouth of the bay (4341¢10¢¢ N, The morphological variability in the genus Ceratium often 719¢00¢¢ E) and is not sheltered from the wind (Nival and exacerbates problems of nomenclature for infraspecific taxa. Corre 1976). The depth of sampling site is 86 m. At Point B, Thereby, an unofficial nomenclature was proposed, defined as physical, chemical, and biological parameters of the water a ‘‘para-taxonomic designation […] on the margin of the column (temperature, chl a, salinity, and main nutrient Linnaean nomenclature’’ by Sournia (1966, p. 1983). The concentrations) were determined weekly using a Seabird monograph of Sournia (1967) includes the description of SBE25 CTD (conductivity–temperature–density; Sea-Bird Elec- varieties, and transitional forms between these varieties, tronics Inc., Bellevue, Washington, USA) for vertical profiles which he characterized as showing a thermal preference and Niskin bottle water samples at six depths (75, 50, 30, 20, 10, (psychrophilic or thermophilic), and forms whose occurrence and 0 m). For statistical analysis, we used minimal, maximal, appears to be independent of the water temperature. For and integrated T (temperature), with integrated T as follows: Z example, the species Ceratium candelabrum is divided into two 1 varieties, C. candelabrum var. candelabrum (type species) and T ¼ Tdz z C. candelabrum var. depressum, and three intermediate forms, C. candelabrum ‘‘candelabrum-depressum,’’ C. candelabrum ‘‘cande- where z is the vertical axis. labrum > depressum’’ (nearer to the variety candelabrum), and A monthly phytoplankton sampling was conducted from C. candelabrum ‘‘depressum > candelabrum’’ (nearer to the variety December 2001 to December 2003. A 0–80 m sampling in depressum). In our study, the infraspecific taxa were distin- double oblique angle was performed with a custom-made guished using this nomenclature, except for C. fusus because conical phytoplankton net (53 lm mesh size, 54 cm diameter the descriptions of its infraspecific taxa vary considerably in the and 280 cm length). The volume filtered by the net was literature (Gourret 1883, Jørgensen 1911, 1920, Tre´gouboff measured with a TSK mechanical flowmeter (Tsurumi-Seiki Co. and Rose 1957a,b, Sournia 1967). Ltd., Yokohama, Japan). The volume sampled depended on The abundance of the rest of microphytoplankton, mainly the intensity of current. The sample was split once or twice diatoms, naked dinoflagellates, and silicoflagellates, was using a Motoda splitter (Motoda 1959), and the aliquot was estimated by integrating data based on Niskin water samples then fixed with acid Lugol’s solution (Throndsen 1978) to a at the six depths monitored at Point B between 0 and 80 m. 2% final concentration. The sample was finally divided into The six samples were mixed in relative proportions to the three replicates to reduce the bias resulting from manual corresponding water column to obtain a single sample per homogenization before counting. Although most phytoplank- date integrating the water column between 0 and 80 m, ton studies based on a net sampling are limited to a qualitative which allows the comparison with net samples. The samples approach, a quantitative analysis is possible when the filtered were concentrated by using the U¨ termohl method (Hasle water volume and the collected water volume are known. Thus, 1978) with an inverted microscope (Axiovert 35; Carl we could estimate the in situ abundance of each taxon in each Zeiss AG. Oberkochen, Germany) at ·200 and ·400 magni- sample. Furthermore, Ceratium cells are covered by cellulosic fication. thecal plates, which resist the water pressure in the net. With Together with the phytoplankton sampling, zooplanktonic cell lengths ranging from 80 to 1200 lm and mean cingular organisms were collected at the same site. Copepods and diameter of 50 lm (Sournia 1986), the loss of cells can be salps were sampled respectively by using a custom-made WP2 considered as negligible when using a 53 lm mesh-size net. net (200 lm mesh size) and a regent net (680 lm mesh size) Organisms were enumerated in several 100 lLor1mL by towing in a double oblique angle between 0 and 80 m calibrated slides. depth. A TSK mechanical flowmeter was also used to determine the volume filtered by the nets. Zooplanktonic organisms were preserved in formaldehyde (4% final concentration). The samples collected were divided into subs- amples with a Motoda splitter (Motoda 1959), and organisms were counted using a dissecting microscope (SZH-ILLD, Olympus Opticals Co. Ltd., Tokyo, Japan). An aggregate zooplankton predation rate was estimated. This predation pressure was considered as the product of abundance of each predator taxa and specific clearance rates taken from the literature (for copepods: Mauchline 1998; for salps: Deibel 1982, Mullin 1983, Madin and Cetta 1984, Andersen 1985). Methodological requirements for biodiversity indices. Studies of the biodiversity of thecate dinoflagellates are often based on the analysis of a relatively small sample volume (e.g., 4 L for Halim 1960) or an unspecified volume. Hence, we needed to estimate the minimal sample volume for a good estimation of the richness, the diversity, and the regularity of Ceratium Fig. 1. Sampling location Point B in the Villefranche Bay, France, Ligurian Sea, northwest Mediterranean. species. These parameters were thus cumulated as a function of MEDITERRANEAN CERATIUM DIVERSITY 1151

Table 1. Species and infraspecific taxa of the genus Table 1. Continued Ceratium in the Villefranche Bay. C. pentagonum var. robustum (Cleve) Jørg. C. arietinum var. arietinum Cleve C. pentagonum var. tenerum Jørg. C. arietinum var. gracilentum (Jørg.) Sournia C. pentagonum Gourret ‘‘robustum-tenerum’’ C. arietinum Cleve ‘‘arietinum-gracilentum’’ C. pentagonum Gourret ‘‘robustum > tenerum’’ C. azoricum Cleve C. petersii Steem. Niels. C. belone Cleve C. platycorne var. platycorne Daday C. candelabrum var. candelabrum (Ehrenb.) F. Stein C. platycorne var. compressum (Gran) Jørg. C. candelabrum var. depressum (C. H. G. Pouchet) Jørg. C. platycorne Daday ‘‘platycorne-compressum’’ C. candelabrum (Ehrenb.) F. Stein ‘‘candelabrum-depressum’’ C. platycorne Daday ‘‘compressum > platycorne’’ C. candelabrum (Ehrenb.) F. Stein ‘‘candelabrum > depressum’’ C. porrectum (G. Karst.) Jørg. C. candelabrum (Ehrenb.) F. Stein ‘‘depressum > candelabrum’’ C. praelongum (Lemmerm.) Kof. ex Jørg. C. carriense var. carriense Gourret C. ranipes Cleve C. carriense var. volans (Cleve) Jørg. C. schroeteri Schro¨d. C. carriense Gourret ‘‘carriense-volans’’ C. setaceum Jørg. C. concilians Jørg. C. symmetricum var. symmetricum Pavill. C. contortum var. contortum (Gourret) Cleve C. symmetricum var. coarctatum (Pavill.) H. W. Graham et Bronik. C. contortum var. karstenii (Pavill.) Sournia C. symmetricum var. orthoceras (Jørg.) H. W. Graham et Bronik. C. contortum var. longinum (G. Karst.) Sournia C. teres Kof. C. contortum var. robustum (G. Karst.) Sournia C. trichoceros (Ehrenb.) Kof. C. contortum (Gourret) Cleve ‘‘contortum-karstenii’’ C. tripos f. hiemale Paulsen C. contortum (Gourret) Cleve ‘‘contortum-robustum’’ C. tripos var. atlanticum (Ostenfeld) Paulsen C. contortum (Gourret) Cleve ‘‘longinum-karstenii’’ C. tripos var. atlanticum f. neglectum (Ostenfeld) Paulsen C. contrarium (Gourret) Pavill. C. tripos var. ponticum Jørg. C. declinatum f. brachiatum Jørg. C. tripos var. pulchellum (Schro¨d.) J. Lo´pez C. declinatum f. declinatum (G. Karst.) Jørg. C. tripos (O. F. Mu¨ll.) Nitzsch ‘‘atlanticum-pulchellum’’ C. declinatum f. normale Jørg. C. tripos (O. F. Mu¨ll.) Nitzsch ‘‘atlanticum > pulchellum’’ C. declinatum var. majus Jørg. C. vultur f. sumatranum G. Karst. C. digitatum F. Schu¨tt C. vultur f. vultur Cleve C. euarcuatum Jørg. C. extensum (Gourret) Cleve No. species: 43. No. infraspecific taxa: 97. C. falcatiforme Jørg. C. falcatum (Kof.) Jørg. C. furca f. corpulentum Jørg. C. furca var. eugrammum (Ehrenb.) J. Schiller the volume of sample analyzed to determine the effect of C. furca var. furca (Ehrenb.) Clarap. et J. Lachm. counting effort. C. furca (Ehrenb.) Clarap. et J. Lachm. ‘‘furca-eugrammum’’ Calculation and estimation of biodiversity indices. Biodiversity C. furca (Ehrenb.) Clarap. et J. Lachm. ‘‘furca > eugrammum’’ was estimated for three replicates as taxonomic richnessP C. furca (Ehrenb.) Clarap. et J. Lachm. ‘‘eugrammum > furca’’ (S = number of taxa), Shannon’s diversity index (H ¢ =– pi C. fusus (Ehrenb.) Dujard. log2 pi,, where pi = ni ⁄ N, ni = number of individuals of one C. gibberum var. dispar (C. H. G. Pouchet) Sournia taxon, and N = total number of individuals), and Pielou’s C. gibberum var. subaequale Jørg. regularity index (J ¢ = H’ ⁄ log2 S). Richness and Shannon’s C. gravidum Gourret index are respectively biased by the sampling effort and by the C. hexacanthum f. hiemale Pavill. sample size (Dallot 1998). In many cases, values of these indices C. hexacanthum f. pavillardii Rampi are underestimated; especially richness (Carpentier and Lepeˆ- C. hexacanthum f. spirale (Kof.) J. Schiller tre 1999), so we used the nonparametric jackknife 1 method C. hexacanthum var. aestuarium (Schro¨d.) Jørg. (Manly 1991) to obtain an estimated value that partially C. hexacanthum var. contortum Lemmerm. corrects this bias for each indicator. For richness, jackknife C. hexacanthum var. hexacanthum Gourret ⁄ C. horridum f. claviger Kof. 1=SO+{r1(n–1) n}, where SO is the observed taxonomic C. horridum var. horridum (Cleve) Gran richness, n the number of replicates, and r1 the number of C. horridum var. buceros (O. Zacharias) Sournia taxa occurring in one single replicate. For diversity and C. horridum (Cleve) Gran ‘‘horridum-buceros’’ regularity, jackknife 1 = SFi ⁄ n, with Fi = nSt–(n–1)Sti–1, where C. horridum (Cleve) Gran ‘‘horridum > buceros’’ St is the estimation of the indicator for the n replicates, and C. horridum (Cleve) Gran ‘‘buceros > horridum’’ Sti–1 the estimation of the indicator for the n–1 replicates. This C. inflatum (Kof.) Jørg. method appears to be a good intermediate choice in terms of C. limulus (Gourret ex C. H. G. Pouchet) Gourret bias and accuracy, according to Carpentier and Lepeˆtre (1999). C. longipes (J. W. Bailey) Gran Moreover, these estimations were calculated for a cumulated C. longirostrum Gourret abundance of 100 cells per replicate (i.e., a total of 300 cells C. longissimum (Schro¨d.) Kof. per sample). Magurran (2004) recommended abundance C. macroceros var. macroceros (Ehrenb.) Vanho¨ffen values between 200 and 500 individuals for diversity calcula- C. macroceros var. gallicum (Kof.) Sournia tion. Finally, specific dominance was considered by the C. macroceros (Ehrenb.) Vanhoffen ‘‘macroceros-gallicum’’ ¨ dominance index d = 100(n1 + n2) ⁄ N, where n1 and n2 are C. macroceros (Ehrenb.) Vanho¨ffen ‘‘gallicum > macroceros’’ the abundances of the two most important species, and N the C. massiliense f. armatum (G. Karst.) Jørg. total cell concentration (Hulburt 1963). In cases where two C. massiliense var. massiliense (Gourret) Jørg. taxa were codominant, the three most abundant taxa were C. massiliense var. protuberans (G. Karst.) Jørg. considered. C. minutum Jørg. C. paradoxides Cleve Statistical analysis. Correlations between Ceratium abun- C. pavillardii Jørg. dance and biodiversity indices and environmental parameters C. pentagonum f. turgidum (Jørg.) Jørg. were made using the nonparametric Spearman’s test. The variability of infraspecific composition was analyzed using a 1152 ALINA TUNIN-LEY ET AL. multivariate analysis yielding a synthesis of co-occurring factors. Density was mostly temperature-driven so that iso- Correspondence analysis (CA) is an ordination method (Ben- pycnal contours (data not shown) matched the iso- zecri 1973) that has been widely used in the analysis of therms. Nitrate concentrations were the highest and ecological data (Gower 1987). The aim was to describe the total inertia of a multidimensional set of data in a sample of fewer homogeneous in winter between December and dimensions (or axes) that is the best summary of the informa- March–April (Fig. 2b), with maximum values above tion contained in the data. Among the inertia methods, CA 1 lM, and decreased between May and December employs contingency tables and uses a chi-square metric. to reach values under the detection limit in early Starting from the cloud of samples within the space of summer. In July, there was a depletion of nitrates infraspecific taxa, and the infraspecific taxa within the space between 0 and 70 m depth, and an accumulation at of samples, this factorial analysis provides the best possible the bottom of the water column (>0.6 lM). The summary of the kinetics of the infraspecific composition over time. The position of a sample in the multivariate space is period characterized by low nitrate concentrations defined using all the infraspecific taxa. Proximity reflects was longer during the year 2003. These variations in similar taxa proportions, while distance reflects dissimilar ones. temperature and nitrates can explain the seasonal The steps of this method and the different calculations are concentrations of chl a (Fig. 2c). There was an detailed in Legendre and Legendre (2000). Our matrix of data increase in the early spring in March—the concen- ) corresponded to a contingency table with the infraspecific taxa trations reaching 0.3–0.45 lg Æ L 1, located between in columns and the samples in rows. The composite mean months’ coordinates, calculated as the average of the samples 0 and 40 m depth—corresponding to the spring of the same month, were used as the illustrative qualitative bloom. The summer was characterized by a decrease variable to synthesize the average annual cycle. This method in chl a and a strong stratification. In late spring, was applied on our data set with the program SPAD 3.5 (SPAD, there was also a development of autotrophic organ- Paris, France). isms deeper in the water column, between 40 and 70 m. Finally the chl a concentrations were observed RESULTS at this same depth at the end of summer, corresponding to the autumn bloom. The hydrological Hydrological structure. Comparing the 2 years, the conditions at Point B showed a stronger stratifica- temperature varied quite similarly (Fig. 2a). It was tion in 2003. below 14C through the water column between Jan- Methodological examination of sampling effort. Exam- uary and April, which characterizes the winter per- ining changes in diversity metrics as a function of iod. However, starting in May, warming of the sample volume, we determined that there was a sta- surface layer was faster and more marked in 2003 bilization of the infraspecific diversity and regularity compared to 2002. In summer 2003, the maximal at 10–20 L. However, infraspecific richness contin- surface temperature was above 25C, whereas it ued to increase and reached a plateau at a sample reached only 23C during the summer of 2002. volume that we estimated to be 70 L minimum at

Fig. 2. Seasonal variations in temperature (a), chl a (b), and nitrates concentrations (c) in the Villefranche Bay (Point B) from December 2001 to December 2003. MEDITERRANEAN CERATIUM DIVERSITY 1153

Fig. 3. Cumulative richness (a), Shannon’s diversity (b), and Pielou’s regularity (c) as a function of sample volume in 2002. Examples are given at the infra- speciﬁc level for each season (May, July, November, and Febru- ary).

) Point B for the genus Ceratium (Fig. 3; Fig. S1 in abundance in May and June 2002 (24 cell Æ L 1) ) the supplementary material). This pattern was and in March 2003 (13 cell Æ L 1). We encountered observed both for a high number of taxa (February 43 species and 97 infraspecific taxa (varieties and 2002 and 2003) and for poor richness, as in May forms in accordance with the nomenclature of Sour- 2003 when the infraspecific richness was very low nia 1967). During the sampling period, infraspecific because of the presence of only one species, Cera- and specific richness reached the highest values in tium furca, and its varieties. the winter period (Fig. 5a). The lowest richness was Seasonal cycle of the genus Ceratium. Total abun- observed in spring, in particular, in April and May dance (Fig. 4) varied between 0 (August 2003) and 2003 when the community was almost exclusively ) 24 cell Æ L 1 (May 2002) with two periods of low composed of C. furca and its infraspecific taxa. The ) abundance (1–2 cell Æ L 1), in spring (April) and in Shannon’s diversity index calculated using specific autumn (October). Similarly, there was a peak of categories (Fig. 5b) was higher in autumn (maxi- ) mum of 4.1 bit Æ ind 1 in October 2002), whereas it ) decreased in spring (minimum of 0.3 bit Æ ind 1 in June 2003). Using infraspecific categories, the minimum diversity also occurred in spring (minimum of ) 2.0 bit Æ ind 1 in April 2003), but there was no distinct seasonal maximum because diversity was not significantly different between winter, summer, and ) fall (maximum of 4.80 bit Æ ind 1 in August 2003). The Pielou’s regularity index (Fig. 5c), which ranges between 0 (no regularity, dominance of one taxon) and 1 (total regularity, no dominance of any taxon), was quite high during the period of study, with a minimum of 0.1 in May 2003 and a maximum of 0.8 in October 2002. Finally, a distinct decrease of all three indices was observed both at specific and infraspecific levels between April and June 2003. The annual cycle was dominated by two species, C. fusus and C. furca (Fig. 6). At least one of the two was present each month of the annual cycle. Ceratium ) Fig. 4. Total abundance (cell Æ m 3) of phytoplankton of the fusus was the most represented species in the winter genus Ceratium from December 2001 to December 2003. Error period, whereas C. furca varieties were dominant bar represents SD calculated from the three replicates. during the spring, especially between April and June 1154 ALINA TUNIN-LEY ET AL.

Fig. 5. Estimated richness (a), Shannon’s diversity (b), and Pielou’s regularity (c) of the genus Ceratium by the jackknife method, at speciﬁc and infraspe- ciﬁc levels, from December 2001 to December 2003. Error bar represents SD calculated from the three replicates.

Fig. 6. Speciﬁc dominance index (%) and dominant species of the genus Ceratium from Decem- ber 2001 to December 2003 in the Villefranche Bay. The ﬁrst species and the second (and third) one are respectively the two (three) most common species observed.

2003 when two taxa of C. furca constituted from distribution of the taxa and the stations in the 71% to 81% of the total of Ceratium cells. Other factorial plane (27.5% and 17.2%). The third important species were C. concilians, C. declinatum, factor was also explanatory (13.6%), but it did not C. extensum, C. massiliense, and C. pentagonum. The represent any obvious ecological meaning in the association C. fusus ⁄ C. extensum seemed to charac- analysis. The projection of the taxa (Fig. 7a) terize the fall period (October and November). showed those which contributed the most to the Ceratium furca, C. fusus, and C. candelabrum occur- position of the two most meaningful (i.e., red throughout the study period. None of most explanatory) factors. Varieties of C. furca con- the infraspecific taxa exhibited such a perennial tributed at 54.8% to the first factor, whereas six occurrence. other taxa, three of which belong to the species The results of the factorial CA (Fig. 7) pro- C. pentagonum, explained 48.9% of factor 2. jected the infraspecific composition of each station The group of C. furca was opposite to a group of in a factorial plane. We retained the first two many taxa whose single contribution to factor 1 factors (axes) as the most explanatory of the was low. MEDITERRANEAN CERATIUM DIVERSITY 1155

The structure of the data could be ecologically explained when stations and the illustrative variable (months) were projected in the same plane (Fig. 7b). Considering the coordinates of the data on axis 1, the winter months September to February (with negative coordinates) were opposed to the spring period, from April to June (positive coordinates). On axis 2, a group of two months with negative coordinates defined a short summer period (July and August). In contrast, the month of March had a positive y-coordinate and a contribution of up to 50% to axis 2. In addition to the characterization of the annual cycle, this representation associated the presence of C. furca and its varieties with spring, and the month of March with the six more explica- tive taxa. In the same way, winter was linked to the presence of numerous taxa. The distance between the months illustrated the importance of changes in the composition of species and infraspecific taxa. Thus, the month of March could be distinguished from other months in terms of infraspecific composition. Influence of temperature. No direct correlation linked Ceratium abundance and diversity to water temperature (maximal or water-column integrated) using the nonparametric Spearman’s correlation method. However, this finding does not mean that water temperature did not directly affect individual species or subspecific taxa; some indeed displayed a seasonal occurrence, whereas others seemed to be perennial. Thus, at the infraspecific level, water temperature could explain the alternation of the different taxa within a species. For example, among species commonly encountered in our samples, C. candelabrum exhibited such a pattern (Fig. 8a). The C. candelabrum var. candelabrum appeared in winter between November and January and disappeared in April or June. Appearance seemed to follow water temperature, conforming to its description as a ‘‘psychrophile’’ (Sournia 1967). The C. candelabrum var. depressum, described as a thermophile by Sournia (1967), while present year- round, was more abundant in terms of relative percentage in June and August. Two species, C. macroceros and C. pentagonum, showed a similar seasonal alternation of varieties. In other species some infraspecific taxa were only somewhat seasonal. Thus, among taxa recorded in each annual cycle, some occurred from September to April (C. arietinum var. arietinum, C. azoricum, C. candelabrum var. candelabrum, C. digitatum, C. falcatiforme, C. horridum var. horridum, C. horridum ‘‘horridum > buceros,’’ C. longissimum, C. pentagonum var. robustum, Fig. 7. Results of the correspondence analysis (CA) on the C. platycorne var. compressum, C. platycorne ‘‘compres- infraspecific abundances of Ceratium taxa, from December 2001 sum > platycorne,’’ C. teres, C. tripos var. atlanticum f. to December 2003. The projection on the factorial plane of all neglectum) and could be considered roughly as win- the infraspecific taxa (circles) indicates labeled items corresponding to the taxa that contribute the most to the two first factors ter taxa. Other taxa seemed to prefer summer con- (a). On the same factorial plane is plotted months of the mean ditions (C. massiliense var. massiliense, C. massiliense annual cycle (black triangles) as illustrative variables (b). The size var. protuberans, C. pentagonum f. turgidum, C. pentago- of items corresponds to the quality of the representation of each num var. tenerum) since their maximal abundances item in the plane. 1156 ALINA TUNIN-LEY ET AL.

Fig. 8. Temporal changes in abundance of three Ceratium species [C. candelabrum (a), C. horridum (b), and C. massiliense (c)] in relation to microphytoplankton abundance from bottle sampling (d) and integrated and surface water temperature (e) from December 2001 to Decem- ber 2003 in the Villefranche Bay.

were observed from May to September; however, The different species and their infraspecific taxa they were never totally absent. The taxa that are cited above are illustrated in Figure S2 (see the sup- not cited above did not follow a distinct seasonal plementary material), reproduced from Jørgensen pattern. (1911, 1920) and Sournia (1967). We considered in more detail shifts in Ceratium Influence of competition and predation. The abun- species, which appear to be composed of several dance of microphytoplankton, based on hydrologi- infraspecific taxa. For example, in the species cal bottle samples, showed two peaks in March and C. massiliense (Fig. 8b), the winter infraspecific taxon April in the bay of Villefranche (Fig. 8d), corre- C. massiliense f. armatum was observed from January to sponding to diatom blooms responsible for the March and disappeared in April, but it was absent in depletion of nutrients in spring. This event, which December 2001 and 2002, while the temperature was likely represented a period of competition for nutri- not significantly different between December and ents, coincided with a decrease in the diversity of January. In the same way, the psychrophilic variety of the genus Ceratium. A microphytoplankton autumn the species C. horridum (Fig. 8c) was observed from bloom also occurred in October 2002 (127,287 ) January to April but also in August 2003, the warmest cells Æ L 1) but was absent the next year. It seemed month of the year, and in December 2003, whereas it there was no relation to the infraspecific variability was absent in December in 2001 and 2002. Thus, in the genus Ceratium in this case. No bloom of these varieties occur in different thermal conditions, toxic phytoplanktonic species was recorded during and other factors probably affect their abundance. the study period. MEDITERRANEAN CERATIUM DIVERSITY 1157

We also examined shifts in the abundance and well estimated for small volumes (approximately infraspecific diversity of the genus Ceratium with 20 L in the Villefranche Bay), taxonomic richness temporal changes in estimated predation pressure was strongly underestimated for the same effort of from two zooplanktonic groups, copepods and salps, analysis. Our results indicate that an estimate of spe- which are known to be important grazers during cies richness requires examination of Ceratium cells spring in the Villefranche Bay. In spring 2002, a collected in at least 70 L of seawater in the Villef- slight decrease in Ceratium diversity occurred with a ranche Bay. This amount can only be obtained bloom of copepods, presumably accompanied by using net sampling. Samples from a few liters will high copepod predation pressure, whereas salp pre- give a good estimation of the Shannon’s and Pie- dation was negligible (Fig. 9). In 2002, copepods lou’s indices, but not of the species richness of Cera- were dominated by species of the genus Clausocal- tium. Likewise, investigators studying only 10 to anus and Oithona similis. In contrast, in 2003 the 100 mL by the U¨ termohl method of sedimentation spring maxima in zooplankton consisted of an might have a good estimation of the diversity indi- unusually high abundance of Calanus helgolandicus ces for a portion of the microphytoplankton (e.g., likely corresponding to a very high predation pres- diatoms, naked dinoflagellates, or silicoflagellates), ) ) sure (231 mL Æ m 3 Æ h 1). Similarly, in June 2003, but certainly not of the richness and the abundance the predation pressure of salps, dominated by the of the genus Ceratium (and probably of all the blastozoid form of Thalia democratica was 30-fold armored dinoflagellates). We recommend that a higher compared to June 2002. This peak salp per- minimal sample volume be established as a first step iod coincided with the critical drop-off of Ceratium in estimating the biodiversity of the genus Ceratium, diversity previously mentioned. The impact of pre- which will vary from area to area. dation by large herbivorous copepods and salps on The genus Ceratium is certainly the most frequent Ceratium diversity seemed to be high. However, the one in our samples and one of the most studied abundance of Ceratium showed a different pattern: marine dinoflagellates in the Mediterranean. At the ) it strongly increased in May 2002 to 24 cell Æ L 1, infraspecific level, it includes 120 reliable taxa, 85 whereas it exhibited low levels in spring 2003 uncertain, and many synonyms (Sournia 1986). In ) (1.3 cell Æ L 1). the Mediterranean, this genus seems to be particularly diverse (Jørgensen 1911, 1920). This conclusion was supported by our results. We found a DISCUSSION higher diversity than that reported in a biogeo- This study, based on net samples, revealed the graphical study that mentioned 30 species of the necessity of analyzing a large volume of water to genus Ceratium in the northwest Mediterranean and determine biodiversity parameters for the genus revealed a biodiversity comparable to tropical areas Ceratium. While the diversity and the regularity were (Dodge and Marshall 1994). However, 52 species of

Fig. 9. Effect of the predation pressure on the genus Ceratium: infraspecific Shannon’s diversity index (a) and abundance (b) of the genus Ceratium in relation to estimated predation pressure applied by herbivorous copepods (c) and salps (d) from December 2001 to December 2003 in the Villefranche Bay. 1158 ALINA TUNIN-LEY ET AL. the genus Ceratium were reported in the Ligurian that were characterized by a particular infraspecific Sea based on literature records in the main sub- composition. Several abiotic or biotic factors could basins of the Mediterranean Sea (Go´mez 2003). It have affected community structure. As noted previ- should be noted that equivalent or slightly higher ously, different authors have emphasized the tem- species richness, consisting of nearly the same set of perature preference of different species and species, was described for the Gulf of Marseilles infraspecific varieties (Sournia 1967, Dodge and (Travers 1975, Travers and Travers 1975). However, Marshall 1994). The seasonal appearance of differ- several taxa were noted as uncertain and are not ent taxa of the genus Ceratium suggests a relation- considered as valid by Sournia (1967). Moreover, ship to temperature. However, some of our results the Gulf of Marseilles benefits from the important are in contradiction to the relationships postulated inputs of the Rhone River and thus corresponds to in the biogeography study of Dodge and Marshall a nutrient-rich environment, contrasting with the (1994). Thus, we characterized two strictly winter relatively poorer waters of the Villefranche Bay, species, C. falcatiforme and C. digitatum, which were although both sites are close to each other. not observed by these authors when water tempera- We observed a maximal abundance in May 2002, ture was below 20C and 25C, respectively. The ) with values (24 cells Æ L 1) 100-fold lower than other winter taxa we collected are within the mini- those observed in the same location in May 1998 mal range of water temperature reported by Dodge (Go´mez and Gorsky 2003) and 24-fold lower than and Marshall (1994), who did not consider distribu- those reported in May 1973 (Rassoulzadegan 1979). tions of infraspecific taxa. Among the species they However, these earlier reports studied concentra- described with a minimal temperature range of tions at a single depth, rather than our average 14C–15C, nine (C. contortum var. karstenii, C. cont- water-column concentrations. A record in the Black rarium, C. declinatum, C. gravidum, C. limulus, C. long- Sea mentioned maximal abundances of 7735 irostrum, C. pavillardii, C. ranipes, C. vultur f. vultur) ) cells Æ L 1 for C. fusus alone (Mikaelyan and Zavyal- were present in February or March in the Villefran- ova 1999). Thus, our values are not high, and they che Bay, although water temperature was below are far from the 1000- to 10,000-fold higher abun- 14C. Yet many of species theoretically absent in dances mentioned in temperate red tides (Weiler water <20C(C. euarcuatum, C. falcatum, C. horridum 1980, Nielsen 1991). It is difficult to directly com- var. buceros, C. inflatum, C. paradoxides) and in water pare the maximal values at a specific depth reported <25C(C. concilians, C. contortum var. longinum, by others with our water-column average values. C. macroceros var. gallicum, C. vultur f. sumatranum) Nevertheless, the differences are large, so it is worth were recorded in winter in Villefranche. These dif- considering whether patchiness—that is, a high ver- ferences can be explained by the sampling methods tical heterogeneity in Ceratium distribution—could used in the studies compiled by Dodge and Marshall explain these differences. Here, we found concen- (1994). For example, some data are from continu- ) trations that exceeded 5 cells Æ L 1 during 56% of ous plankton recorder (CPR). This sampling covers the sampling period. This threshold is equivalent to large sea surfaces, but it does not consider the verti- maximal abundances that we measured (i.e., 6 cal dimension since the CPR collects at a constant ) cells Æ L 1) in the South Pacific gyre in the samples depth close to the surface. Thus, taxa that usually from the BIOSOPE cruise in September–October develop in deeper waters cannot be collected. The 2004, whereas it varied respectively from 15 to problem of insufficient sample size could also ) 19 cells Æ L 1 in the upwelling and the high nutrient induce bias in the studies based on bottle sampling. low chlorophyll zone (HNLC). Another study With regard to the relationships postulated by ) observed Ceratium concentrations of 2 cells Æ L 1 Sournia (1967), only some of the taxa corresponded or less in the Pacific central gyre in January 1983 to the seasonal preference description, especially in (Matrai 1986). Thus, the Ligurian Sea is character- the winter taxa group. The projection of the maxi- ized by a Ceratium assemblage close to tropical or mal, minimal, and integrated temperatures on the subtropical assemblages in terms of richness, but factorial plane of CA did not reveal a significant with numerical abundances higher than those effect on the specific and infraspecific composition. observed in tropical areas, probably related to a This finding could easily be explained with our sam- more nutrient-rich environment. pling method, which allowed an accurate estimation In the Villefranche Bay, the annual cycle of the of biodiversity parameters and integrated sampling genus Ceratium was characterized by a seasonal trend through the water column but did not consider the with a marked contrast between the spring period effect of the thermocline in summer. Since the (minimum richness, diversity, and regularity) and water temperature under the thermocline was often the winter period (highest taxonomic richness). close to the winter temperatures, it is possible that Regularity followed the same seasonal pattern as the some species described as psychrophile could grow diversity index. Therefore, the diversity was then in deep water during the stratified period, from more influenced by the distribution of the taxa than June to November. by their numbers. The CA revealed a continuous Relationships between varieties of a single and repeated annual cycle with biological seasons Ceratium species and abiotic factors could be MEDITERRANEAN CERATIUM DIVERSITY 1159 questionable. For instance, one could say that ther- autotrophic organisms increases. Therefore, while mophilic varieties have morphologies adapted to strictly autotrophic forms may be favored in winter, warm water (i.e., a less viscous environment than mixotrophy or heterotrophy could be favored in cold water, inducing a faster sedimentation). To spring as a result of competition with diatoms for reduce sinking velocity, phytoplankton cells with a nutrients. In addition, at the end of the spring larger surface-to-volume ratio (S ⁄ V), thereby bloom, heterotrophy in the genus Ceratium could be increasing drag resistance, could be favored. We possible by senescent algae exudation of dissolved noted this adaptation in our study for several spe- organic matter (DOM; Nagata 2000). Direct osmo- cies, among them, C. candelabrum and C. horridum. trophy has not been demonstrated in the genus They all had more long and delicate extensions Ceratium, but it is conceivable that DOM indirectly (horns) in summer varieties. But, as noted by benefits taxa of the genus Ceratium. For example, Hutchinson (1967), ‘‘the problem of the rate of DOM is assimilated by bacteria, which are either sinking of a body in a viscous medium is one of directly consumed by ciliates or via flagellates, which unexpected complexity’’ (p. 256). Sournia (1982) are themselves ingested by ciliates, the main prey of was also disappointed in his synthesis concerning some Ceratium (Smalley et al. 1999). form and function in marine phytoplankton, With regard to C. fusus, the ratio between the emphasizing the multiple relationships of the S ⁄ V heterotrophic and autotrophic populations at the ratio with, for example, nutrient absorption, light end of the summer may depend on nutrient flux availability, or grazing pressure. If we consider a from the intermediate layers to the upper layers relationship between morphology of varieties of a (Mikaelyan and Zavyalova 1999). The ability to single species of Ceratium and ecological factors, change trophic strategy could thus explain the con- those taxa should be defined as ecomorphs (Sour- sistent presence of this species. Moreover, the annual nia 1967). But another hypothesis is to regard those cycle was characterized by the almost exclusive pres- varieties as cryptic—or more accurately, pseudocryp- ence of C. furca between April and June 2003, as well tic—species with different thermic preferences. For as during the high-abundance period of the genus example, the nanoplanktonic group of coccolitho- Ceratium, observed in May and June 2002. High con- phorids is known to present many pseudocryptic centrations of dinoflagellates, with a remarkable species, distinct species differing only in fine-scale dominance of C. furca, have been described several morphological variations, previously described as times for this period in the bay of Villefranche varieties or subspecies (Saéz et al. 2003). The exis- (Halim 1960, Go´mez and Gorsky 2003). This phe- tence of cryptic species has been reported in fresh- nomenon could result from the mixotrophic capacity water diatoms and is strongly suggested in marine of C. furca, which would be favored during a period ones (Orsini et al. 2004, Lundholm et al. 2006). In when diatoms exude DOM. Indeed, C. furca is able dinoflagellates, Crypthecodinium cohnii Seligo is to feed on a great variety of prey (small dinoflagel- known to be a sibling species complex (Beam et al. lates, ciliates, flagellates, amoebae) with a preference 1993); cryptic species have also been genetically for small ciliates (Smalley et al. 1999). Moreover, a identified in the coastal genus Scrippsiella (Montres- positive correlation between its ingestion rate (het- or et al. 2003) and in Alexandrium tamarense (M. Le- erotrophic strategy) and the concentration in prey bour) Balech (John et al. 2003). Morphometric and dissolved organic nitrogen have been shown analysis associated with molecular phylogeny studies (Smalley and Coats 2002). Finally, this hypothesis is is required to discriminate ecomorphs from pseudo- supported by the work of Sournia (1967) who not cryptic species in Ceratium. Nonetheless, at the spe- only described the thermic preference of the varie- cific or infraspecific level, some taxa of the genus ties but also noticed that the taxa were oligotrophic Ceratium were clearly linked to water temperature, or oligophotic—namely, preferring poor environ- while others, although previously described as show- ments, where heterotrophy is a necessity—or eupho- ing a thermal preference, seemed to be indepen- tic, where autotrophy is possible. However, other dent of this abiotic factor. authors, using epifluorescence, noted that every spe- A possible alternative to temperature control is cies of Ceratium collected (29 species) contained chl competition. This factor is particularly interesting a (Lessard and Swift 1986). We have come to similar among Ceratium species as their trophic role appears conclusions. Using epifluorescence microscopy, all quite variable. They can be autotrophic, as they do the living cells of some 26 Ceratium species collected possess chloroplasts; heterotrophic, by means of at different depths, from 300 m depth to the surface, phagocytosis; and mixotrophic (Sournia 1986, Smal- in May and June 2005, exhibited the presence of ley and Coats 2002). The succession of Ceratium taxa chl a. These results suggest that if heterotrophy exists could reflect diversity in trophic strategies. Effec- in this genus, mixotrophy would be predominant. tively, the period from January to March corre- Another study noted that inclusion bodies were com- sponded to cold temperature conditions, high mon in oceanic Ceratium containing chloroplasts in nutrient concentrations, and, consequently, low the main part of the cells, but the authors could not competition for nutrients. Starting in April, because prove that inclusion bodies were food vacuoles of the spring diatom bloom, competition among (Chang and Carpenter 1994). 1160 ALINA TUNIN-LEY ET AL.

The diversity and the abundance of the genus grazing pressure, and maybe other factors. This Ceratium could also depend on top-down control. does not mean that Ceratium species and infraspe- Our results suggested that the spring bloom of salps cific taxa could not be used as biological indicators dominated by T. democratica and the high abun- of climate change. First, certain Ceratium taxa are dance of copepod Ca. helgolandicus influenced the clearly linked to water temperature and have abundance and the diversity of the genus Ceratium already been proposed as indicators for water in the bay of Villefranche, whereas copepods below masses (Sanchez et al. 2000), current regimes 2 mm in length did not. In fact, in the northwest (Dowidar 1973), and climate change (Dodge and Mediterranean, the salp population increases during Marshall 1994). The annual cycle showed recurrent spring and occasionally to very high concentrations, events such as the spring C. furca dominance or so that they dominate the zooplankton (Meńard the late winter maximal richness, which could be et al. 1994, Fernex et al. 1996). Their individual used for dating biological seasons and for identify- grazing rates are mostly higher than individual grazing potential shifts. Moreover, the presence of typi- ing rates of other herbivores (Madin and Purcell cally rare warm-temperate to tropical taxa may be 1992), and the particle retention spectra ranges indicators for warming since they should develop from 2 lm to 1 mm (Harbison and McAlister 1979, greater numbers in warmer conditions. Second, we Kremer and Madin 1992, Madin and Deibel 1998). also indicated that Ceratium assemblages vary with The size-range of length in the genus Ceratium is competition, particularly with the presence of dia- from 80 lm to 1.2 mm (Sournia 1986). Recently, tom blooms. One possible effect of global warming Vargas and Madin (2004) presented evidence that is a negative impact on diatom abundance due to T. democratica naturally fed on autotrophic dinofla- a higher water stratification inducing a decrease in gellates. nutrient availability (Bopp et al. 2005). Third, since In addition to salps, many other organisms may predators could also affect Ceratium assemblages, feed on Ceratium species. Crustaceans, which often the nature of consumers is important. Shifts in the dominate zooplankton communities, presumably ratio of crustaceans to gelatinous zooplankton is could have a significant grazing impact on Ceratium possible (see consequences of anthropic impacts cells. However, it seems that only the largest cope- in the Black Sea, Kideys and Romanova 2001, pod species and cladocerans are able to feed on Oguz et al. 2001) and could have a very impor- them (Nielsen 1991). Among the different copepod tant impact on phytoplankton structure. Finally, species in the North Sea, only females of the large Ceratium species are easily sampled and recognized; copepod Ca. helgolandicus had a relevant grazing they have been studied for a long time, and an effect on C. furca during the egg production period abundant literature is available, in particular histor- (Jansen et al. 2006). Elbra¨chter (1973) indicated ical data for the Mediterranean Sea. The availabil- that the genus Ceratium was ingested by both cope- ity of historical data is sparse for other easily pods and ciliates, with a preference for recently recognized phytoplanktonic groups. Among other divided smaller cells. microphytoplankton taxa, the well-studied and With regard to other dinoflagellates, in labora- abundant diatoms are still providing challenges in tory cultures, another mixotrophic dinoflagellate terms of species determinations. In conclusion, ingested cells of C. tripos and C. lineatum by phagocy- taxa of Ceratium are sensitive to temperature, com- tosis (Hansen et al. 2000). In addition, Protoperidini- petitors, and predators, and shifts in community um was able to feed on C. furca (Olseng et al. 2002) composition could be considered as an integrator while the size in this genus is from 25 to 275 lm of the possible biotic and abiotic effects of global (Sournia 1986). Thus, cells of the same size or even changes. smaller can be predators of the genus Ceratium. Besides, the dominance of C. furca during spring We thank anonymous reviewers and the associate editor for their very useful suggestions, which led to significant could have also resulted from a selection by preda- improvements in our manuscript. Hydrological data were tors in favor of this species and its varieties. Indeed, provided by Service d’Observation en Milieu Littoral, INSU- copepods are known to be size-selective feeders CNRS, Observatoire Oceánologique de Villefranche-sur-mer. (Wilson 1973), whereas salps seem to filter water We thank the sailors of the Observatoire Oceánologique de without any particle selection (Kremer and Madin Villefranche, J.-Y. Carval and J.-L. Prevost, for sampling at 1992). However, it is more likely that the domi- Point B. Financial support was provided by INSU of the CNRS through the project CNRS-INSU ATI #01N50 ⁄ 0388 nance of C. furca is due to its mixotrophic ability ‘Effet du Mesozooplancton sur la Diversite´ des Comparti- rather than preferential grazing on other Ceratium ments Phyto- et Microzooplanctonique d’une Zone Coˆtie`re species. Me´diterraneénne.’ This work was also supported by the Mar- Recent multivariate models suggest that bottom- BEF European Network of Excellence ‘Marine Biodiversity up and top-down controls may have interdepen- and Ecosystem Functioning’ in the framework of the pro- dent effects on biological diversity and ecosystem gram MARPLAN for European integration of marine micro- functioning (Worm et al. 2002). It is likely that the plankton research. Ph.D. scholarship was offered by ‘‘Ministe`re de l’Education Nationale, de l’Enseignement diversity of the genus Ceratium depends simulta- Supe´rieur et de la Recherche.’’ We also thank the BIOSOPE neously on water temperature, nutrient availability, MEDITERRANEAN CERATIUM DIVERSITY 1161 group (PROOF-JGOFS-France) for providing Pacific samples. Hutchinson, G. E. 1967. A Treatise on Limnology, Vol. II. Wiley and We would like to thank Dr. John Dolan for English correc- Sons, New York, 1115 pp. tions. Jansen, S., Wexels Riser, C., Wassman, P. & Bathmann, U. 2006. Copepod feeding behaviour and egg production during a dinoflagellate bloom in the North Sea. Harmful Algae 5:102–12. Andersen, V. 1985. Filtration and ingestion rates of Salpa fusiformis John, U., Fensome, R. A. & Medlin, L. K. 2003. The application of a Cuvier (Tunicata: Thaliacea): effects of size, individual weight molecular clock based on molecular sequences and the fossil and algal concentration. J. Exp. Mar. Biol. Ecol. 87:13–29. record to explain the biogeographic distributions within the Beam, C. A., Preparata, R. M., Himes, M. & Nanney, D. L. 1993. Alexandrium tamarense ‘‘species complex’’ (Dinophyceae). Mol. Ribosomal RNA sequencing of members of the Crypthecodinium Biol. Evol. 20:1015–27. cohnii (Dinophyceae) species complex; comparison with solu- Jørgensen, E. 1911. Die Ceratien. Eine kurze Monographie des ble enzyme studies. J. Eukaryot. Microbiol. 40:660–7. Gattung Ceratium Schrank. Int. Rev. Hydrobiol. 4:1–124. Benzecri, J. P. 1973. L’analyse des Donneés. II. L’Analyse des Corre- Jørgensen, E. 1920. Mediterranean Ceratia. Rep. Dan. Oceanogr. Ex- spondances. Dunod, Paris, 619 pp. ped. Mediter. 1:1–110. Bockstahler, K. R. & Coats, D. W. 1993. Spatial and temporal Kideys, A. E. & Romanova, Z. 2001. Distribution of gelatinous aspects of mixotrophy in Chesapeake Bay dinoflagellates. macrozooplankton in the southern Black Sea during 1996– J. Eukaryot. Microbiol. 40:49–60. 1999. Mar. Biol. 139:535–47. Bopp, L., Aumont, O., Cadule, P., Alvain, S. & Gehlen, M. 2005. Kremer, P. & Madin, L. P. 1992. Particle retention efficiency of Response of diatoms distribution to global warming and salps. J. Plankton Res. 14:1009–15. potential implications: a global model study. Geophys. Res. Lett. Legendre, P. & Legendre, L. 2000. Numerical Ecology, 2nd ed. 32:L19606. doi: 10.1029/2005GL023653. Elsevier Science BV, Amsterdam, 853 pp. Carpentier, P. & Lepeˆtre, A. 1999. Robustesse de quelques indices Lessard, E. J. & Swift, E. 1986. Dinoflagellates from the North de diversiteá` l’ećhantillonnage. Oceanis 25:435–55. Atlantic classified as phototrophic or heterotrophic by epiflu- Chang, J. & Carpenter, E. J. 1994. Active growth of the oceanic orescence microscopy. J. Plankton Res. 8:1209–15. dinoflagellate Ceratium teres in the Caribbean and Sargasso seas Lundholm, N., Moestrup, Ø., Kotaki, Y., Hoef-Emden, K., estimated by cell cycle analysis. J. Phycol. 30:375–81. Scholin, C. & Miller, P. 2006. Inter- and intraspecific variation Dallot, S. 1998. Sampling properties of biodiversity indices. Oceanis of the Pseudo-nitzschia delicatissima complex (Bacillariophy- 24:89–105. ceae) illustrated by rRNA probes, morphological data and Deibel, D. 1982. Laboratory-measured grazing and ingestion rates phylogenetic analyses. J. Phycol. 42:464. of the salp, Thalia democratica Forskal, and the doliolid, Madin, L. P. & Cetta, C. M. 1984. The use of gut fluorescence Dolioletta gegenbauri Uljanin (Tunicata, Thaliacea). J. Plankton to estimate grazing by oceanic salps. J. Plankton Res. 6:475– Res. 4:189–201. 92. Dodge, J. D. 1982. Marine Dinoflagellates of the British Isles. Her Madin, L. P. & Deibel, D. 1998. Feeding and energetics of Thalia- Majesty’s Stationery Office, London, 303 pp. ceae. In Bone, Q. [Ed.] The Biology of Pelagic Tunicates. Oxford Dodge, J. D. 1993. Biogeography of the planktonic dinoflagellate University Press, Oxford, UK, pp. 81–103. Ceratium in the western Pacific. Korean J. Phycol. 8:109–19. Madin, L. P. & Purcell, J. E. 1992. Feeding, metabolism, and growth Dodge, J. D. & Marshall, H. G. 1994. Biogeographic analysis of the of Cyclosalpa bakeri in the subarctic Pacific. Limnol. Oceanogr. armoured planktonic dinoflagellate Ceratium in the North 37:1236–51. Atlantic and adjacent seas. J. Phycol. 30:905–22. Magurran, A. E. 2004. Measuring Biological Diversity. Blackwell Dowidar, N. M. 1973. Distribution and ecology of Ceratium egyptia- Publishing, Oxford, UK, 256 pp. cum Halim and its validity as an indicator of the current regime Manly, B. F. 1991. Randomization and Monte Carlo Methods in Biology. in the Suez Canal. J. Mar. Biol. Assoc. India 15:335–44. Chapman and Hall, London, 292 pp. Elbra¨chter, M. 1973. Population dynamics of Ceratium in coastal Matrai, P. 1986. The distribution of the dinoflagellate Ceratium in waters of the Kiel Bay. Oikos 15:43–8. relation to environmental factors along 28N in the eastern Fernex, F. E., Braconnot, J. C., Dallot, S. & Boisson, M. 1996. Is North Pacific. J. Plankton Res. 8:105–18. ammonification rate in marine sediment related to plankton Mauchline, J. 1998. The biology of calanoid copepods. Adv. Mar. composition and abundances? A time series study in Villef- Biol. 33:1–710. ranche Bay (NW Mediterranean) Estuar. Coast. Shelf Sci. Meńard, F., Dallot, S., Thomas, G. & Braconnot, J. C. 1994. Tem- 43:359–71. poral fluctuations of two Mediterranean salp populations from Go´mez, F. 2003. Checklist of Mediterranean free-living dinoflagel- 1967 to 1990. Analysis of the influence of environmental lates. Bot. Mar. 46:215–42. variables using a Markov chain model. Mar. Ecol. Prog. Ser. Go´mez, F. & Gorsky, G. 2003. Microplankton annual cycles in the 104:139–52. Bay of Villefranche, Ligurian Sea, NW Mediterranean. Mikaelyan, A. S. & Zavyalova, T. A. 1999. Vertical distribution of J. Plankton Res. 25:323–39. heterotrophic phytoplankton in the Black Sea during Gourret, P. 1883. Sur les pe´ridiniens du golfe de Marseille. Ann. the summer period. Oceanology (Russian Acad. Sci.) 39:893– Mus. Hist. Nat. Marseille 1:1–114. 902. Gower, J. C. 1987. Introduction to ordination techniques. Devel- Montresor, M., Sgrosso, S., Procaccini, G. & Kooistra, W. 2003. opments in numerical ecology. NATO ASI Ser. Ser. G Ecol. Sci. Intraspecific diversity in Scrippsiella trochoidea (Dinophyceae): 14:3–64. evidence for cryptic species. Phycologia 42:56–70. Halim, Y. 1960. Etude quantitative et qualitative du cycle ećologi- Motoda, S. 1959. Devices of simple plankton apparatus. Mem. Fac. que des dinoflagelle´s dans les eaux de Villefranche-sur-Mer. Fish. Hokkaido Univ. 7:73–94. Ann. Inst. Oceanogr. Monaco 38:123–232. Mullin, M. M. 1983. In situ measurements of filtering rates of salps, Hansen, P. J., Skovgaard, A., Glud, R. N. & Stoecker, D. K. 2000. Thalia democratica, on phytoplankton and bacteria. J. Plankton Physiology of the mixotrophic dinoflagellate Fragilidium sub- Res. 5:279–88. globusum. II. Effects of time scale and prey concentration on Nagata, T. 2000. Production mechanisms of dissolved organic photosynthetic performance. Mar. Ecol. Prog. Ser. 201:137–46. matter. In Kirchman, D. L. [Ed.] Microbial Ecology of the Oceans. Harbison, G. R. & McAlister, V. L. 1979. The filter-feeding rates and Wiley-Liss, New York, pp. 121–52. particle retention efficiencies of three species of Cyclosalpa Nielsen, T. G. 1991. Contribution of zooplankton grazing to the (Tunicata, Thaliacea). Limnol. Oceanogr. 24:875–92. decline of a Ceratium bloom. Limnol. Oceanogr. 36:1091–106. Hasle, G. R. 1978. The inverted-microscope method. In Sournia, A. Nival, P. & Corre, M. C. 1976. Variations annuelles des caracte´ris- [Ed.] Phytoplankton Manual. UNESCO, Paris, pp. 88–96. tiques hydrologiques de surface dans la baie de Villefranche- Hulburt, E. M. 1963. The diversity of phytoplanktonic populations sur-Mer. Ann. Inst. Oceanogr. Paris 52:57–78. in oceanic, coastal, and estuarine regions. J. Mar. Res. 21:81–93. 1162 ALINA TUNIN-LEY ET AL.

Oguz, T., Ducklow, H. W., Purcell, J. E. & Malanotte-Rizzoli, P. Smalley, G. W., Coats, D. W. & Stoecker, D. K. 2003. Feeding in the 2001. Modeling the response of top-down control exerted by mixotrophic dinoflagellate Ceratium furca is influenced by gelatinous carnivores on the Black Sea pelagic food web. intracellular nutrient concentrations. Mar. Ecol. Prog. Ser. J. Geophys. Res. 106:4543–64. 262:137–51. Okolodkov, Y. B. 1996. Net phytoplankton from the Barents Sea Sournia, A. 1966. Sur la variabilite´ infraspećifique du genre Cera- and Svalbard waters collected on the cruise of the R ⁄ V tium (pe´ridinien planctonique) en milieu marin. CR Acad. Sci. ‘‘Geolog Fersman’’ in July-September 1992, with emphasis on Paris 263:1980–3. the Ceratium species as biological indicators of the Atlantic Sournia, A. 1967. Le genre Ceratium (Pe´ridinien planctonique) waters. Bot. J. Russian Acad. Sci. 81:1–9. dans le canal du Mozambique. Contribution a` une re´vision Olseng, C. D., Naustvol, L. J. & Paasche, E. 2002. Grazing by the mondiale. Vie Milieu A: Biol. Mar. 18(1968):375–500. heterotrophic dinoflagellate Protoperidinium steinii on a Cera- Sournia, A. 1982. Form and function in marine phytoplankton. tium bloom. Mar. Ecol. Prog. Ser. 225:161–7. Biol. Rev. Camb. Philos. Soc. 57:347–94. Orsini, L., Procaccini, G., Sarno, D. & Montresor, M. 2004. Multiple Sournia, A. 1986. Classe des Dinophyceés. In Sournia, A. [Ed.] Atlas rDNA ITS-types in the diatom Pseudo-nitzschia delicatissima du Phytoplancton Marin. Centre National de la Recherche Sci- (Bacillariophyceae) and their relative abundances across a entifique, Paris, pp. 26–98. spring bloom in the Gulf of Naples. Mar. Ecol. Prog. Ser. Steidinger, K. A. & Tangen, K. 1997. Dinoflagellates. In Tomas, C. 271:87–98. R. [Ed.] Identifying Marine Phytoplankton. Academic Press, San Raine, R., White, M. & Dodge, J. D. 2002. The summer distribution Diego, California, pp. 387–584. of net plankton dinoflagellates and their relation to water Sullivan, J. M. & Swift, E. 1995. Photoenhancement of biolumi- movements in the NE Atlantic Ocean, west of Ireland. nescence capacity in natural and laboratory populations of the J. Plankton Res. 24:1131–47. autotrophic dinoflagellate Ceratium fusus (Ehrenb.) Dujardin. Rassoulzadegan, F. 1979. Cycles annuels de la distribution de J. Geophys. Res. 100:6565–74. diffe´rentes cate´gories de particules du seston et essai d’iden- Throndsen, J. 1978. Preservation and storage. In Sournia, A. [Ed.] tification des principales pousseés phytoplanctoniques dans Phytoplankton Manual. UNESCO, Paris, pp. 69–74. les eaux ne´ritiques de Villefranche-sur-mer. J. Exp. Mar. Biol. Travers, M. 1975. Inventaire des protistes de Marseille et de ses Ecol. 38:41–56. parages. Ann. Inst. Oceánogr. Paris 51:51–75. Saéz, A. G., Probert, I., Geisen, M., Quinn, P., Young, J. R. & Travers, A. & Travers, M. 1975. Catalogue of the microplankton in Medlin, L. K. 2003. Pseudo-cryptic speciation in coccolitho- the Gulf of Marseilles. Int. Rev. Hydrobiol. 60:251–76. phores. Proc. Natl. Acad. Sci. 100:7163–8. Tre´gouboff, G. & Rose, M. 1957a. Manuel de Planctonologie Me´diter- Sanchez, G., Calienes, R. & Zuta, S. 2000. The 1997–98 El Ninõ and raneénne, Tome I. Centre National de la Recherche Scientifique, its effects on the coastal marine ecosystem off Peru. Rep. CA Paris, 587 pp. Coop. Ocean. Fish. 41:62–86. Tre´gouboff, G. & Rose, M. 1957b. Manuel de Planctonologie Me´diter- Sato, H., Greuet, C., Cachon, M. & Cosson, J. 2004. Analysis of the raneénne, Tome II. Centre National de la Recherche Scientifi- contraction of an organelle using its birefringency: the R-fibre que, Paris, 203 pp. of the Ceratium (Dinoflagellates) flagellum. Cell Biol. Int. Vargas, C. A. & Madin, L. P. 2004. Zooplankton feeding ecology: 28:387–96. clearance and ingestion rates of the salps Thalia democratica, Semina, H. J. & Levashova, S. S. 1993. The biogeography of tropical Cyclosalpa affinis and Salpa cylindrica on natural occur- phytoplankton species in the Pacific Ocean. Int. Rev. Hydrobiol. ring particles in the Mid-Atlantic Bight. J. Plankton Res. 26:827– 78:243–62. 33. Skovgaard, A., Hansen, P. J. & Stoecker, D. K. 2000. Physiology of Weiler, C. S. 1980. Population structure and in situ division rates of the mixotrophic dinoflagellate Fragilidium subglobusum. I. Ceratium in oligotrophic waters of the North Pacific central Effects of phagotrophy and irradiance on photosynthesis and gyre. Limnol. Oceanogr. 25:610–9. carbon content. Mar. Ecol. Prog. Ser. 201:129–36. Weiler, C. S. & Eppley, R. W. 1979. Temporal pattern of division in Smalley, G. W. & Coats, D. W. 2002. Ecology of the red-tide dino- the dinoflagellate genus Ceratium and its application to flagellate Ceratium furca: distribution, mixotrophy, and grazing the determination of growth rate. J. Exp. Mar. Biol. Ecol. 39:1– impact on ciliate populations of Chesapeake Bay. J. Eukaryot. 24. Microbiol. 49:63–73. Wilson, D. S. 1973. Food size selection among copepods. Ecology Smalley, G. W., Coats, D. W. & Adam, E. J. 1999. A new method 54:909–14. using microspheres to determine grazing on ciliates by the Worm, B., Lotze, H. K., Hillebrand, H. & Sommer, U. 2002. Con- mixotrophic dinoflagellate Ceratium furca. Aquat. Microb. Ecol. sumer versus resource control of species diversity and ecosys- 17:167–79. tem functioning. Nature 417:848–51. MEDITERRANEAN CERATIUM DIVERSITY 1163

Supplementary Material The following supplementary material is available for this article: Figure. S1. Cumulative richness (a), Shannon’s diversity (b), and Pielou’s regularity (c) as a function of sample volume in 2003. Examples are given at infraspecific level for each season (May, July, November, and February). Figure. S2. Infraspecific taxa of Ceratium candelabrum, Ceratium horridum, and Ceratium massiliense: C. candelabrum var. candelabrum (a), C. candelabrum ‘‘candelabrum > depressum’’ (b), C. candelabrum ‘‘candelabrum-depressum’’ (c), C. candelabrum var. depressum (d), C. horridum var. horridum (e), C. horridum ‘‘horridum-buceros’’ (f), C. horridum ‘‘buceros > horridum’’ (g), C. horridum ‘‘horridum > buceros’’ (h), C. horridum var. buceros (i), C. horridum f. claviger (j), C. massiliense var. protuberans, (k) C. massiliense f. armatum (l), and C. massiliense var. massiliense (m). Figures (j) and (k) are reproduced from Jørgensen (1911). Figure (m) is reproduced with permission from Jørgensen (1920). All others figures are reproduced with permission from Sournia (1967). Scale bars, 50 lm. This material is available as part of the online article from: http://www.blackwell-synergy.com/ doi/abs/10.1111/j.1529-8817.2007.00417.x. (This link will take you to the article abstract.) Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.